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. 2019 Nov;19(4):355–367. doi: 10.17305/bjbms.2019.4227

FIGURE 2.

FIGURE 2

Silencing of F-box/WD repeat-containing protein 7 (FBXW7) significantly decreased the population of CD133-positive cells and reversed c-MYC expression in PC9 gefitinib-resistant persisters (GRPs). (A) FBXW7 expression was knocked down in PC9-GRPs with short interfering RNA (siRNA) followed by gefitinib treatment for 72 h and examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Two specific siRNAs and one non-specific control siRNA were used, and representative data from the siRNA experiment are shown. Average values for cells treated with the control siRNA were set to 1. Average values were calculated from triplicate experiments. Error bars, SD. **p < 0.01. (B, left) FBXW7 protein expression knocked down with siRNA in PC9-GRPs cells after gefitinib treatment for 72 h was analyzed by immunofluorescence. Nuclei were stained with DAPI. (C, left) c-MYC expression was analyzed by immunofluorescence after silencing FBXW7 followed by gefitinib treatment for 72 h in PC9-GRPs cells. DAPI (blue) was used to stain nuclei. (B, right and C, right) The ratio of FBXW7-positive cells (B, right) or c-MYC-positive cells (C, right) in PC9-GRPs cells after silencing FBXW7. Average values were calculated from five fields in each sample. **p < 0.01, scale bar indicates 50 µm. (D) Knockdown of FBXW7 expression with siFBXW7 followed by gefitinib treatment for 72 h in PC9-GRPs cells significantly suppressed CD133 expression as determined by qRT-PCR. Average values and SDs were calculated from triplicate samples. Average values for cells treated with the control siRNA were set to 1. **p < 0.01. (E, left) PC9 cells were cultured on slides of Labo-Tek chamber, and FBXW7 expression was silenced with siFBXW7. Cells were treated with 1 µM gefitinib for 72 h. CD133-positive GRPs were significantly decreased. DAPI (blue) was used to stain nuclei. (E, right) The ratio of CD133-positive cells in PC9-GRPs after silencing FBXW7 and followed by gefitinib treatment for 72 h. Average values were calculated from five fields in each sample **p< 0.01, scale bar indicates 50 µm.